Folates and classical antifolates such as MTX.sup.1 are converted intracellularly to poly(.gamma.-glutamyl) metabolites by the enzyme folylpolyglutamate synthetase. EQU PteGlu.sub.n+ ATP+L-Glu.fwdarw.PteGlu.sub.n+1+ ADP+P.sub.i
Since it is now known that folylpolyglutamates are essential to the proper functioning of folate metabolism, and antifolylpolyglutamates are implicated in the cytotoxic action of classical antifolates such as MTX, folylpolyglutamate synthetase has become an important enzyme for study in folate biochemistry and biochemical pharmacology. In this regard, the specificity of this enzyme for pteroyl and L-glutamate substrates has been extensively investigated. For pteroyl substrates, the structure of the heterocyclic component can vary considerably, but a terminal L-glutamate residue has been shown to be absolutely required for substrate activity in all reports to this time. Specificity for the incoming amino acid is strict, but not absolute. L-homocysteic acid and D,L-ervthro- or D,L -threo-4-fluoroglutamate can all serve as efficient alternate substrates, but in each case incorporation causes chain termination. Chain termination by the 4-fluoroglutamate diastereomers demonstrates the stringent specificity for L-glutamate at the .gamma.-g1utamyl acceptor site.
F.sub.2 Glu is a potent, concentration-dependent inhibitor of poly(.gamma.-glutamylation) using [.sup.3 H]Glu and either methotrexate (4-NH.sub.2 -10-CH.sub.3 PteGlu) or tetrahydrofolate as substrates. Applicants have determined that F.sub.2 Glu acts as an alternate substrate, but in contrast to the previously characterized alternate substrate 4-fluoroglutamate (McGuire and Coward, J. Biol. Chem. 260: 6747 (1985)), it did not terminate polyglutamate chain elongation. Instead, F.sub.2 Glu promotes chain elongation. Thus, synthesis of products from [.sup.3 H]methotrexate containing 1 and 2 additional amino acid residues occurs at a substantially higher rate in the presence of F.sub.2 Glu when compared to identical reactions in the presence of Glu; this is more pronounced for the product containing 2 additional residues. The increased rate of addition is not solely a function of ligating F.sub.2 Glu to the internal Glu or to a previously incorporated F.sub.2 Glu, since ligation of Glu to 4-NH.sub.2 -10-CH.sub.3 PtGlu-.gamma.-(3,3-difluoroglutamate) is also enhanced. These results are consistent with F.sub.2 Glu enhancing the synthesis of poly(.gamma.-g1utamate) metabolites at the level of either the incoming amino acid (glutamate analog) or the .gamma.-glutamyl acceptor species. F.sub.2 Glu is thus the first glutamate analog which enhances chain elongation catalyzed by folylpolyglutamate synthetase.